Recent improvements in DNA sequencing techniques have sought to meet the increasing demands of large scale sequencing. Increasingly, methods in which the template nucleic acid molecules are attached to a solid surface are being developed (see, for example, U.S. Pat. No. 5,302,509 and U.S. Pat. No. 5,547,839). Such methods dispense with the need for an electrophoretic separation step and, with he use of optical detection technologies (see, for example, Nie et al. Annu. Rev. Biophys. Biomol. Struct. 1997, 26: 567–96), aim to allow sequencing information at the level of a single molecule to be obtained. This has the further potential for multiple samples to be analysed simultaneously.
One example of such methods is Base Addition Sequencing Scheme (BASS) (see, for example, Metzker et al., Nucleic Acids Res 1994, Vol.22, No.20; p. 4259–4267). BASS is a method involving the incorporation of nucleotide analogues which have been modified so as to comprise a blocking group which terminates DNA synthesis. A primer is annealed to a template bound to a solid support and sequence data obtained by repetitive cycles of incorporation of modified nucleotides. At each cycle, the incorporated base is identified in situ before being deprotected to remove the blocking group and allow the next cycle of DNA synthesis.
Methods such as BASS rely on the use of nucleotide analogues that possess polymerase enzyme blocking (or terminator) groups at the 3′ hydroxyl position of the sugar on the nucleotide. Typically, the blocking group is a combined terminator and label/reporter moiety such that the incorporated nucleotide can be detected while the bulky label or reporter moiety itself fulfils the role of blocking a polymerase from any further DNA synthesis. Conveniently, as the terminator group is also the reporter moiety, a single reaction allows simultaneous removal of both functions thus allowing subsequent DNA synthesis and for incorporation of the next base to be read.
In order to allow subsequent rounds of DNA synthesis, these polymerase enzyme blocking groups are, typically, attached to the nucleotide via a linking group in such a way that they can be removed. However, conventional sequencing strategies require high temperatures of cycling (typically approximately 95° C. or above) which are associated with pH changes in the reaction mixture. Such conditions can cause reactivity of certain chemical bonds. Accordingly, the coupling methods for attaching blocking and labelling groups to nucleotides which have been used to date have focused on using those linking groups which can withstand changes in chemical conditions (such as temperature and pH). For example, the blocking and label groups can be attached via photosensitive linkage groups and thus cleavable by light irradiation (i.e. photochemical means, see, for example, WO 93/05183) or via chemical means.
However, the use of known nucleotide analogues suffers from a number of disadvantages.
Firstly, by attaching the bulky reporter moiety in the 3′ position of the nucleotide, the ability of the DNA polymerase to recognise or tolerate the nucleotide is reduced. Currently known nucleotide terminators are incorporated by polymerases with an efficiency which fails to approach 97%. In addition to being poorly incorporated, modified nucleotides may be inactive (i.e. not incorporated), inhibitory (i.e. inhibit DNA synthesis) or may result in an alteration of the polymerase enzyme fidelity.
Secondly, the known methods of removing the terminator groups require repeated insult by reactive chemicals or irradiation which can result in damage to the template DNA strand through reactions such as base transformation, crosslinking, or depurination.
Any one of, or a combination of, these effects will result in a reduced accuracy in the sequence data obtained and, in particular, a decreased signal-to-noise ratio will be found on detection. Moreover, this means that the amount of sequence data that can be obtained from successive rounds of enzyme incorporation and cleavage is limited. For example, if a combined error of approximately 3% in incorporation and cleavage were to accumulate, the result would be that sequence could only be obtained from 5 bases or fewer of the template DNA before the decreased signal to noise ratio made further sequencing impractical.
Accordingly, there is a need for improved nucleotide analogues. Such analogues may have one or more of the following attributes: tolerated by polymerases; stable during the polymerization phase; and blocking groups can be removed efficiently under conditions which minimise damage to the template strand or template-primer complex. Preferably, the improved analogues display more than one of these features and most preferably they display all of these features.
It is thus an object of the invention to provide a nucleotide analogue to which blocking and reporter moieties are attached at separate positions of the nucleotide. It is another object of the invention to provide a nucleotide analogue to which blocking and reporter moieties are attached via linking groups which are enzyme-cleavable groups. Such latter nucleotide analogues are most suitable for using in sequencing reactions which involve an isothermic reaction and therefore do not involve exposure of the nucleotide analogues to high temperatures and to undesirable variations in chemical conditions. Under the conditions of suitable sequencing reactions, including array-based sequencing technologies (such as BASS), enzyme-cleavable groups will be essentially stable. The use of enzyme-cleavable linking groups removes the need for harsh, template-damaging treatments to remove the blocking and reporter moieties.